The World Health Organization estimated that at the end of 2012 over 35 million individuals were infected with HIV-1 (Human Immunodeficiency Virus). Due to the advancements in highly active antiretroviral (HAART) therapy the number of AIDS-related deaths has been reduced and individuals are living longer. Despite successes with HAART, opportunistic infections remain one of major causes of death in AIDS patients. Among the most prevalent is the protozoal parasite pathogen, Cryptosporidium. Cryptosporidiosis is one of the AIDS- defining illnesses characterized by a severe chronic diarrhea for which there are currently no effective therapies and among the most frequent pathogens causing diarrheal diseases in developing countries. Efforts during the previous grant period have focused on a unique bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) found only in protozoal pathogens as a possible molecular target for inhibitor design. These proof of concept studies in Cryptosporidium hominis (Ch)TS-DHFR have identified unique species specific and allosteric/non-active target regions in this bifunctional enzymes and mutational analysis and mechanistic studies have validated these sites as essential for catalytic function. The PIs lab and their collaborators have developed a distinctive and successful computationally and mechanistically guided approach for the discovery of new inhibitors of ChTS-DHFR. This partnership of computational and detailed experimental methodologies is a unique strategy that builds optimal physiochemical and pharmacological parameters into the design. These molecules are then experimentally tested and the design iteratively refined through mechanistic, structural and cellular evaluation. Molecular docking and virtual screening coupled with structural analyses have discovered novel inhibitors of CH TS-DHFR including some with nanomolar potency and anti-Cryptosporidial activity in cell culture. The current grant period will focus on lead optimization and a novel nanotechnology strategy for parasite drug delivery to develop new therapies to treat Cryptosporidial infections.